Device and method for controlling the optical power in a microscope

ABSTRACT

An apparatus for controlling optical power in a microscope includes a measuring device for measuring the optical power, and a control unit for controlling a high-frequency source as a function of the measured optical power so as to achieve a selectable level of the optical power. The microscope includes a source providing light along an illumination beam path to a sample, a detector receiving detection light lead along a detection beam path from the sample, and an acousto-optical or electro-optical element located in the illumination beam path and driven by the high-frequency source.

[0001] Priority is claimed to U.S. provisional application 60/544,207and to German patent application DE 103 24 331.3, the subject matters ofboth of which are hereby incorporated by reference herein.

[0002] The present invention relates to a device and a method forcontrolling the optical power in a microscope, in particular, in aconfocal laser scanning microscope, including at least one light source,an illumination beam path leading the light to a sample, a detector, adetection beam path leading the detection light from the sample to thedetector, and further including an acousto-optical or electro-opticalelement in the illumination beam path.

BACKGROUND

[0003] Devices and methods for controlling the optical power in amicroscope are known in the field. In the devices of the type described,fast acousto-optical or electro-optical elements are generally used toadjust the optical power of an illuminating light beam in a nearlyinfinitely variable and spectrally selective manner. The opticalelements used here are primarily AOTF (acousto-optical tunable filter)crystals which allow the optical power of a laser used as a light sourcein a microscope to be controlled for each wavelength. To this end,generally, an RF frequency corresponding to the desired laser wavelengthand a corresponding amplitude of the RF wave are applied to the AOTFcrystal via a control unit.

[0004] The operating principle of an AOTF crystal is based on the factthat, for example, the RF frequency applied to the crystal acts as anoptical grating for a laser beam incident perpendicular to the RFfrequency, allowing the incident laser light to be diffracted into thefirst order maximum in a nearly completely collinear manner, and thus tobe provided as an illuminating light beam. By linear superposition ofdifferent RF frequencies, laser light of different wavelengths can becollinearly diffracted and picked off with different intensities at theAOTF.

[0005] A crucial parameter of acousto-optical elements is the soundvelocity, i.e., the velocity at which the RF wave applied to the crystalpropagates in the crystal. A change in the sound velocity results in achange in the diffraction efficiency of the crystal, i.e., the laserwavelength diffracted with maximum intensity is shifted in frequency.

[0006] A device of the type described is known, in particular, fromGerman Patent DE 198 27 140 C2. The laser scanning microscope describedtherein has also an AOTF crystal provided in the input laser beam pathfor spectrally selective adjustment of the optical power of theilluminating light beam. Since the sound velocity in the AOTF crystal istemperature-dependent, a temperature sensor is provided in the vicinityof the crystal, the temperature sensor registering the temperature as ameasuring signal. To maintain the optical illumination power constant,it is proposed there as a first measure to maintain the AOTF crystal ata constant temperature using heating control means. As an alternativemeasure, it is proposed to control the AOTF frequency via a control unitas a function of the detected temperature to thereby correct a change inthe optical power of the illuminating light beam resulting from thetemperature change.

[0007] To carry put such a correction, calibration curves are neededfrom which can be derived the relationship between a change in thetemperature of the crystal and a resulting change in the diffractionefficiency or a shift in frequency of the optimally diffracted laserwavelength.

[0008] This method has the problem that, on the one hand, it only relieson the correctness of the underlying calibration curve and, on the otherhand, that measuring errors in the calibration curves will propagate.Moreover, its use in practice is extremely inflexible because a newcalibration curve must first be generated for each illuminationwavelength.

[0009] A further problem is that the actual crystal temperature can onlybe measured with a delay in time. Due to the dimensions of the crystal,the time constant is typically several minutes. However, due to theabsorption of the RF power in the crystal, the temperature can alsochange on a shorter time scale so that inaccuracies may creep in thismanner as well.

SUMMARY OF THE INVENTION

[0010] It is therefore an object of the present invention to provide adevice and a method for controlling the optical power in a microscope,in particular, in confocal laser scanning microscope, whereby theoptical power of the illuminating light beam of a microscope can beeasily adjusted with high precision in a spectrally selective mannerand, in particular, maintained constant.

[0011] The present invention provides an apparatus for controllingoptical power in a microscope, where the microscope includes at leastone light source configured to provide light along an illumination beampath to a sample; a detector configured to receive detection light leadalong a detection beam path from the sample; and an acousto-optical orelectro-optical element disposed in the illumination beam path andconfigured to be driven by a high-frequency source. The apparatusincludes a measuring device configured to measure the optical power; anda control unit configured to control the high-frequency source as afunction of the measured optical power so as to achieve a selectablelevel of the optical power.

[0012] In accordance with the present invention, it was discovered,first of all, that control of the optical power as a function of thetemperature prevailing at the acousto-optical element is highlyerror-prone. In a next step, it was found that these errors can beavoided by a more direct procedure, i.e., by detecting the optical poweritself instead of the temperature as an indirect parameter. Theacousto-optical or electro-optical element can be operated by a controlunit as a function of the measured optical power in such a manner thatthe optical power assumes a desired value. This allows the optical powerto be controlled with high precision.

[0013] From a design standpoint, the measuring device could, in generalterms, be placed at any position in illumination beam path.

[0014] Advantageously, the measuring device could be placed between ascanning optical system and a tube optical system in the plane of anintermediate image. In this connection, the measuring device could beable to be rotated into the area of the actual image field in the planeof the intermediate image. However, it is also conceivable to place themeasuring device outside the actual image field at the edge of theintermediate image, it being possible for such a configuration to be afixed configuration.

[0015] Particularly precise control of the optical power is possiblewhen placing the measuring device directly upstream of the sample. Bypositioning the measuring device in this manner, it is possible toeliminate nearly all sources of error that occur along the illuminationbeam path and which can affect the optical power on the sample. Theerror sources to be mentioned in this connection are, in particular, thebeam splitters or the deflection mirrors, which are generally providedwith vapor-deposited, polarization-dependent coatings. Specifically, theoptical power can thus be stabilized to below 1% change. It is alsopossible, in particular, to compensate for the non-linear properties ofa piezoelectric transducer that is used to couple the RF wave into theacousto-optical crystal.

[0016] When examining transparent samples, it is also conceivable toplace the measuring device downstream of the sample.

[0017] If the samples to be examined are insensitive, and therequirements placed on the measurement are primarily to maintainconstant the optical power incident on the detector, then the measuringdevice could even be placed in the detection beam path.

[0018] The measuring device can have detection means that are used tomeasure the optical power. Specifically, the detection means can bephotodiodes. Especially when measuring short-time fluctuations of theoptical power, it is suitable to use monitoring diodes.

[0019] In addition to the detection means, the measuring device couldalso have reference patterns. These reference patterns could, forexample, be in the form of a grating or lines and used for calibration,in particular, for linearity calibration.

[0020] Alternatively, or in addition to placing a measuring device inthe illumination beam path, it would also be possible to provide ameasuring device outside the illuminating beam path, the illuminatingand/or the detection light beam being reflected onto the measuringdevice via an optical means for preferably permanently reflecting out areference beam.

[0021] The microscope may include a beam combiner which allows light ofdifferent wavelengths, i.e. in particular the light of several differentlasers, to be combined into an illuminating light beam in a manner knownper se. To select specific wavelengths and to adjust the optical powerin a nearly infinitely variable and spectrally selective manner, itwould be possible to place the acousto-optical or electro-opticalelement downstream of the beam combiner. As already described, theoptical power can be controlled in a spectrally selective manner as afunction of the optical power measured by the measuring device.

[0022] The acousto-optical element used can be, in particular, an AOTF(acousto-optical tunable filter), an AOBS (acousto-optical beamsplitter), or an AOM (acousto-optical modulator).

[0023] The electro-optical element used could be an EOM (electro-opticalmodulator). In this case, the voltage applied to the crystal can becontrolled directly.

[0024] Particularly advantageously, the RF frequency and/or the RFamplitude of the high-frequency and/or voltage source driving theacousto-optical or electro-optical element could be adjustable in anearly infinitely variable manner. If the measured optical powerdeviates from the desired value, the optical power could then becorrected with high precision in a spectrally selective manner bycontrolling the RF frequency and/or the RF amplitude.

[0025] In addition to the above-described acousto-optical andelectro-optical element, which is placed downstream of the beam combinerto select specific wavelengths in the illuminating light beam, it wouldalso be possible to provide further acousto-optical and/orelectro-optical elements. These elements could also be controllable as afunction of the optical power measured by the measuring device. Theseadditional acousto-optical and/or electro-optical elements could be usedin a well-known manner to spatially separate the illuminating light beamand the detection light beam. With regard to the relevant backgroundart, reference is made to Unexamined German Laid-Open Patent ApplicationDE 101 37 155.

[0026] The present invention also provides a method for controlling theoptical power in a microscope, in particular, in confocal laser scanningmicroscope. According to the method, the optical power is measured usinga measuring device, and a high-frequency and/or voltage source drivingthe acousto-optical or electro-optical element is controlled by acontrol unit as a function of the measured optical power in such amanner that the optical power reaches a selectable level.

[0027] Specifically, provision could be made for the high-frequencyand/or voltage source to be controlled in such a manner that the opticalpower incident on the sample remains constant. This is particularlyimportant when examining sensitive samples, i.e., to ensure that thesesamples are not damaged or even destroyed by excessive optical power.However, in special applications, it is also conceivable control thehigh-frequency and/or voltage source in such a manner that the opticalpower passes through a selectable profile.

[0028] As described above in connection with the device according to thepresent invention, the optical power could be measured in anintermediate image in a particularly simple manner. In the intermediateimage, the measurement could be carried out, for example, on aframe-by-frame basis, which is useful especially in the case oflong-term measurements. However, it would also be conceivable to carryout the measurement each time before an image is recorded. For opticalpower measurement, it is advantageous to measure the optical power in aspectrally selective manner so as to be able to control the opticalpower at the acousto-optical and electro-optical elements based on themeasured values in a spectrally selective manner as well.

[0029] Generally, it would be possible to correct minor fluctuations ofthe optical power by changing the RF amplitude of the high-frequencysource that drives the acousto-optical element while maintaining the RFfrequency constant. Given appropriate initial conditions, this should beeasily possible within a temperature range of +/−1° C., in which thepower change is less than 3%.

[0030] If, in the case of larger fluctuations of the optical power,control of the RF amplitude alone is not sufficient, it would bepossible to record a complete intensity-frequency curve to therebydetermine an optimum RF frequency of the high-frequency source drivingthe acousto-optical element. Given an appropriate control and use ofsuitable drivers, the optimum RF frequency can be determined in lessthan a second.

[0031] In the case of weak laser lines, variation of the RF amplitude isproblematic because this amplitude should virtually always be set to100%. In this case, it would be possible to correct the RF frequency assoon as an optical power loss of 1% is measured.

[0032] In the case of an AOBS used to spatially separate theilluminating light beam and the detection light beam, it has turned outto be advantageous to correct only the RF frequency because otherwisethe excitation light component in the detection light beam is increased;it also being possible to, at least partially, compensate for thiseffect using a second downstream subcomponent of the AOBS.

[0033] Instead of using the illuminating light as a direct parameter, itis also conceivable to feed in an additional measuring beam whose poweris always kept constant. This test beam could be provided, for example,by an IR laser diode. The light should not disturb the sample. The testbeam could be guided through the AOTF, for example, aside the actualilluminating light or perpendicularly to the illuminating light. All RFfrequencies are then simultaneously corrected analogously to the RFfrequency of the test beam (according to a previously definedrelationship).

BRIEF DESCRIPTION OF THE DRAWING

[0034] The present invention is elaborated upon below based on anexemplary embodiment with reference to the drawing, in which:

[0035]FIG. 1 shows a schematic representation of a device according tothe present invention for controlling the optical power in a microscope,in particular, in a confocal laser scanning microscope.

DETAILED DESCRIPTION

[0036]FIG. 1 shows a device according to the present invention forcontrolling the optical power in a microscope, the microscope shown inthe Figure being designed as a confocal scanning microscope. Themicroscope includes two lasers 1, 3 whose emitted light beams 5, 7 havedifferent wavelengths and which are combined into an illumination beampath 11 by dichroic beam combiner 9. In addition, the microscope has anacousto-optical element 59, which is designed as an AOTF 61.

[0037] After passage through the AOTF 61, illumination beam path 11proceeds to a deflection mirror 12 and from there to a furtheracousto-optical element 13, which is designed as an AOTF 15. Fromacousto-optical element 13, illumination beam path 11 reaches a beamdeflection device 17 which contains a gimbal-mounted scanning mirror 19and guides the illumination beam path 11 through scanning optical system21, tube optical system 23, and lens 25 and across or through sample 27.

[0038] A measuring device 55 is placed between scanning optical system21 and tube optical system 23 in the plane of the intermediate image,the measuring device having detection means for measuring the opticalpower and/or reference patterns in the form of a grating or lines forcalibration.

[0039] The detection beam path 29 originating from the sample runs inthe opposite direction through scanning optical system 21, tube opticalsystem 23, and lens 25 and reaches acousto-optical element 13 viascanning mirror 19, the acousto-optical element 13 feeding detectionlight beam 29 to a further acousto-optical element 31, which issimilarly designed as an AOTF 33 and used for spectral splitting. Afterpassage through acousto-optical element 31, detection beam path 29reaches detector 39, which is designed as a multiband detector.

[0040] In the drawing, illumination beam path 11 is represented as asolid line, and detection beam path 29 is shown as a dashed line.Illumination pinhole 41 and detection pinhole 43, which are usuallyprovided in a confocal scanning microscope, are schematically drawn forthe sake of completeness. By contrast, a number of optical elements forguiding and shaping the light beams have been omitted for the sake ofimproved clarity. They are sufficiently known to a one skilled in thisfield.

[0041] The acousto-optical element 59 used for selecting the selectedwavelength components of illuminating light beam 11 is designed as anAOTF 61 which is traversed by an acoustic wave. The acoustic wave isgenerated by an electrically driven piezoelectric sound generator 63.Control is via a high-frequency source 57, the high-frequencyelectromagnetic wave being transmitted through a coaxial cable 48. TheRF frequencies are selected such that only the selected wavelengthcomponents enter illumination beam path 11 and reach beam deflectiondevice 17. The remaining components that are not affected by theacoustic excitation are blocked from illumination beam path 11. In thisconnection, the crystal cut and orientation of acousto-optical element59 are selected such that, given the same input direction, differentwavelengths are deflected in the same direction.

[0042] The power of the light of the desired wavelengths in illuminationbeam path 11 can be selected by varying the RF frequency and/or the RFamplitude of the acoustic wave as a function of the optical powerdetected by measuring device 55. If the measured optical power deviatesfrom the desired optical power, then high-frequency source 57 iscontrolled via a control unit 53 in such a manner that the optical powerreaches the desired level again. This procedure can be carried out in acolor-selective fashion for the laser wavelengths contained in theilluminating light beam.

[0043] AOBS 14 is composed of the two further acousto-optical elements13 and 31, which are designed as an AOTF 15 and 33, and driven by afurther high-frequency source 47 and 51, respectively. In thisconnection, the RF frequencies are selected, for example, such that thedetection light components in detection beam path 29 that have thewavelength of the illuminating light are blocked. Acousto-opticalelement 13 is essentially used to separate the illuminating light beamand the detection light beam. After passage through AOTF 15, thedetection light beam has undergone both spectral andpolarization-dependent splitting. Acousto-optical element 31 isessentially used to compensate for the spectral andpolarization-dependent splitting. Both acousto-optical elements 13 and31 can be controlled independently as a function of the optical powerdetected by measuring device 55.

[0044] Finally, it should be noted that the exemplary embodimentdiscussed above serves to illustrate the claimed teaching withoutlimiting it to the exemplary embodiment.

[0045] List of Reference Numerals

[0046]1 laser

[0047]3 laser

[0048]5 emitted light beam

[0049]7 emitted light beam

[0050]9 beam combiner

[0051]11 illumination beam path

[0052]12 deflection mirror

[0053]13 acousto-optical element

[0054]14 AOBS

[0055]15 AOTF

[0056]17 beam deflection device

[0057]19 scanning mirror

[0058]21 scanning optical system

[0059]23 tube optical system

[0060]25 lens

[0061]27 sample

[0062]29 detection beam path

[0063]31 acousto-optical element

[0064]33 AOTF

[0065]39 detector

[0066]41 illumination pinhole

[0067]43 detection pinhole

[0068]45 piezoelectric sound generator

[0069]47 high-frequency and/or voltage source

[0070]48 coaxial cable

[0071]51 high-frequency and/or voltage source

[0072]53 control unit

[0073]55 measuring device

[0074]57 high-frequency and/or voltage source

[0075]59 acousto-optical element

What is claimed is:
 1. An apparatus for controlling optical power in amicroscope, the microscope including: at least one light sourceconfigured to provide light along an illumination beam path to a sample;a detector configured to receive detection light lead along a detectionbeam path from the sample; and an acousto-optical or electro-opticalelement disposed in the illumination beam path and configured to bedriven by a high-frequency source; the apparatus comprising: a measuringdevice configured to measure the optical power; and a control unitconfigured to control the high-frequency source as a function of themeasured optical power so as to achieve a selectable level of theoptical power.
 2. The apparatus as recited in claim 1 wherein themicroscope includes a confocal laser scanning microscope.
 3. Theapparatus as recited in claim 1 wherein the high-frequency sourceincludes a voltage source.
 4. The apparatus as recited in claim 1wherein the measuring device is disposed in the illumination beam path.5. The apparatus as recited in claim 1 wherein the measuring device isdisposed between a scanning optical system and a tube optical system ina plane of an intermediate image of the microscope.
 6. The apparatus asrecited in claim 1 wherein the measuring device is disposed directlyupstream of the sample.
 7. The apparatus as recited in claim 1 whereinthe measuring device is disposed downstream of the sample.
 8. Theapparatus as recited in claim 1 wherein the measuring device is disposedin the detection beam path.
 9. The apparatus as recited in claim 1wherein the measuring device includes a detection device configured tomeasure optical power.
 10. The apparatus as recited in claim 9 whereinthe detection device includes at least one of a photodiode and amonitoring diode.
 11. The apparatus as recited in claim 1 wherein themeasuring device includes at least one reference pattern.
 12. Theapparatus as recited in claim 1 further comprising an optical reflectingdevice configured to reflect out a reference beam.
 13. The apparatus asrecited in claim 12 wherein the optical reflecting device is configuredto permanently reflect out the reference beam.
 14. The apparatus asrecited in claim 1 wherein the acousto-optical or electro-opticalelement is disposed in the illumination beam path downstream of a beamcombiner.
 15. The apparatus as recited in claim 1 wherein theacousto-optical element includes an acousto-optical tunable filter. 16.The apparatus as recited in claim 1 wherein the acousto-optical elementincludes an acousto-optical beam splitter.
 17. The apparatus as recitedin claim 1 wherein the acousto-optical element includes anacousto-optical modulator.
 18. The apparatus as recited in claim 1wherein the electro-optical element includes an electro-opticalmodulator.
 19. The apparatus as recited in claim 1 wherein at least oneof an RF frequency and an RF amplitude of the high-frequency source isadjustable in a nearly infinitely variable manner.
 20. The apparatus asrecited in claim 1 further comprising at least one furtheracousto-optical or electro-optical element controllable as a function ofthe measured optical power.
 21. A method for controlling optical powerin a microscope, comprising: measuring the optical power using ameasuring device; driving an acousto-optical or electro-optical elementof the microscope using a high-frequency source; and controlling thehigh-frequency source using a control unit, the controlling beingperformed as a function of the measured optical power so as to achieve aselectable level of the optical power.
 22. The method as recited inclaim 21 wherein the microscope includes a confocal laser scanningmicroscope.
 23. The method as recited in claim 21 wherein the microscopeincludes: at least one light source configured to provide light along anillumination beam path to a sample; and a detector configured to receivedetection light lead along a detection beam path from the sample. 24.The method as recited in claim 23 wherein the acousto-optical orelectro-optical element is disposed in the illumination beam path. 25.The method as recited in claim 21 wherein the high-frequency sourceincludes a voltage source.
 26. The method as recited in claim 21 whereinthe controlling is performed so that the optical power remains constant.27. The method as recited in claim 21 wherein the controlling isperformed so that the optical power passes through a selectable profile.28. The method as recited in claim 21 wherein the measuring is performedby measuring the optical power in an intermediate image.
 29. The methodas recited in claim 28 wherein the measuring is performed by measuringthe optical power in the intermediate image on a frame-by-frame basis.30. The method as recited in claim 21 wherein the measuring is performedby measuring the optical power each time before an image is recorded.31. The method as recited in claim 21 wherein the measuring is performedby measuring the optical power in a reference beam that is permanentlyreflected out.
 32. The method as recited in claim 21 wherein themeasuring is performed by measuring the optical power in a spectrallyselective manner.
 33. The method as recited in claim 21 furthercomprising correcting minor fluctuations of the optical power bychanging an RF amplitude of the high-frequency source.
 34. The method asrecited in claim 21 further comprising, upon larger fluctuations of theoptical power, recording a complete intensity-frequency curve so as todetermine an optimum RF frequency of the high-frequency source.
 35. Themethod as recited in claim 21 wherein the controlling is performed bycorrecting, upon an occurrence of weak laser lines, an RF frequency ofthe high-frequency source when an optical power loss of the laser lineis 1%.
 36. The method as recited in claim 21 wherein: the microscopefurther includes an acousto-optical beam splitter disposed in adetection beam path; and wherein the controlling is performed bycorrecting an RF frequency of the high-frequency source at theacousto-optical beam splitter.